The role of retinoids such as all-trans retinoic acid (ATRA), 13-cis retinoic acid (13-cis RA) and synthetic retinoic acid (RA) analogs in mediating cell growth and differentiation has generated interest in their pharmacological utility for controlling the treatment of dermatological diseases, such as psoriasis and acne, as well as oncological applications such as chemotherapy and chemoprevention. Significant advances in elucidating the molecular basis of retinoid action now offer the potential for designing RA compounds with improved therapeutic indices.
To date, several receptors for retinoic acid have been identified. These receptors are members of a superfamily of intracellular receptors which function as ligand dependent transcription factors. At present, these receptors have been classified into two subfamilies, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). The classification of these subfamilies is based primarily on differences in amino acid structure, responsiveness to different naturally occurring and synthetic retinoids, and ability to modulate expression of different target genes. Each RAR and RXR subfamily has three distinct isoforms designated RAR.alpha., RAR.beta. and RAR.gamma., and RXR.alpha., RXR.beta. and RXR.gamma.. The discovery of multiple retinoid receptors raises questions of the functional properties of the distinct subfamilies and their isoforms.
Recently, it has been discovered that 9-cis RA is capable of binding to and modulating gene expression via the RARs and RXRs. Heyman et al., Cell, 68:397 (1992). The discovery of this property of 9-cis RA has led to further investigation into the biochemical properties of the RARs and RXRs with naturally occurring RA as well as with synthetic retinoids.
One technique for determining the affinity of RA and synthetic retinoids to RARs and associated proteins is to employ a competitive ligand binding assay using radiolabeled compounds showing RAR activity. See e.g., U.S. Pat. No. 5,196,577. These ligand binding studies require substantial quantities, i.e. greater than 50 milliCuries, of a high specific activity radiolabeled compound (e.g., over 10 Ci/mmol). However, conventional syntheses of radiolabeled retinoids, such as radio-labeled ATRA, using e.g. photoisomerization, is costly and generally cannot yield sufficient quantities of a high specific activity radiolabeled retinoid to effectively conduct ligand binding studies. For example, in H. H. Kaegi et al., J. Labelled Compd. Radiopharm., 18:1099 (1981), reduction of an aldehyde compound with a mixture of lithium borohyride and lithium borotride yielded a of mixture monohyride and monotride intermediates, which were ultimately synthesized to labeled all-trans retinoic acid and all-trans retinyl acetate with an activity of generally less than 3 Ci/mmol. See also, M. I. Dawson et al., J. Labelled Compd. Radiopharm., 33:245 (1993) (photoizomerization to yield .mu.Ci quanties of tritium-labeled 9-cis retinoic acid). Thus, conventional syntheses simply cannot provide sufficient quantities of high activity radiolabeled retinoid stereoisomers, such as 9-cis RA and 9,13-dicis RA. Furthermore, to date, no radiolabeled compounds have been identified that show activity on RXRs, as well as RARs.
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